Expression of the chicken GDNF family receptor α-1 as a marker of spermatogonial stem cells.

The identification, enrichment and subsequent isolation of spermatogonial stem cells (SSCs) are integral to the success of SCC transplants between fertile donor and sterilized recipient males. In birds generally and particularly in chicken, SSC-specific has yet to be identified. The receptor for glial cell-derived neurotrophic factor (GDNF), i.e. GDNF family receptor alpha-1 (GFRα1), has been identified as a potential marker for different mouse spermatogonial subtypes. In the present study, we characterized the chicken cGFRα1 receptor and compared its predicted amino-acid sequence with mouse, rat and human GFRα1 proteins. Using specific polyclonal mouse anti-cGFRα1 serum, a total of 2.8% cells were recognized as cGFRα1-positive among isolated testicular cells recovered from sexually mature cockerels. The percentages of cGFRα1-positive testicular cells with haploid, diploid, tetraploid and SP DNA content were 1.6%, 2.5%, 39.3% and 76.8%, respectively. The presence of cGFRα1 protein on the surfaces of all cells of the seminiferous epithelium was confirmed by immunocytochemical and immunohistochemical analyses. Tissue specificity of cGFRα1 mRNA expression was significantly higher in adult testes compared to brain tissue which itself was several times higher than tissues prepared from the spleen, liver and heart. No expression was observed in muscular tissue. At last, we demonstrated the successful repopulation of sterilized recipient's testes with transplanted cGFRα1-positive donor testicular cells. Recipient males subsequently produced functional heterologous spermatozoa capable of fertilizing an ovum and obtaining chicks with donor cell genotypes.

Identification of various testicular cell populations in pubertal and adult cockerels.

Precise identification of the male germinal stem cell population is important for their practical use in programs dedicated to the integration of exogenous genetic material in testicular tissues. In the present study, our aim was to identify germinal cell populations in the testes of pubertal and adult cockerels based on the detection of the nuclear DNA content by fluorescence-activated cell sorting (FACS) and on the expression of the Dazl and Stra8 genes in single-cell suspensions of testicular tissues. Cells with a tetraploid DNA content (4c) represent a small and equal fraction of the total germinal cell population in both pubertal and adult males. In contrast, the diploid (2c) and haploid (c) subpopulations differ significantly between ages as a consequence of different degrees of sexual maturation. A specific subpopulation of testicular cells, the side-scatter subpopulation of cells, or side population (SP), was identified at the junction between the haploid and diploid cell populations. The percentage of this cell subpopulation differs significantly in pubertal and adult cockerels, accounting for 4.1% and 1.3% of the total cell population, respectively. These four testicular cell populations were also tested for the expression of Dazl and Stra8 genes known to be expressed in premeiotic cells including stem spermatogonia. Both genes were expressed in SP, whereas the expression of either Dazl or Stra8 genes was detected only in the 4c and in the 2c testicular cell subpopulations, respectively. The correlation between the cell ploidy and Dazl/Stra8 expression was the same at both male ages. We conclude that SP cells might represent a subpopulation of germinal cells enriched in stem spermatogonia, which can be of great importance for transgenesis in chicken.

Close linkage of genes encoding receptors for subgroups A and C of avian sarcoma/leucosis virus on chicken chromosome 28.

Avian sarcoma and leucosis viruses (ASLV) are classified into six major subgroups (A to E and J) according to the properties of the viral envelope proteins and the usage of cellular receptors for virus entry. Subgroup A and B receptors are identified molecularly and their genomic positions TVA and TVB are mapped. The subgroup C receptor is unknown, its genomic locus TVC is reported to be genetically linked to TVA, which resides on chicken chromosome 28. In this study, we used two chicken inbred lines that carry different alleles coding for resistance (TVC(R) and sensitivity (TVC(S)) to infection by subgroup C viruses. A backross population of these lines was tested for susceptibility to subgroup C infection and genotyped for markers from chicken chromosome 28. We confirmed the close linkage between TVA and TVC loci. Further, we have described the position of TVC on chromosome 28 relative to markers from the consensus map of the chicken genome.

Intraembryonic avian leukosis virus subgroup C (ALV-C) inoculation producing wasting disease in ducks soon after hatching.

We have studied the pathogenic changes in Khaki Campbell ducks injected in mid embryogenesis with ALV subgroup C virus td daPR-C derived from a molecular clone. The employed duck flock was shown to be highly genetically homogeneous and was controlled for the absence of current infections. Clear symptoms of wasting disease, which appeared since one week post hatching, represented the early consequence of the virus infection. They were manifested by decreased body weight, including clear involution of thymic tissue and pronounced anaemia. Microscopically, thymuses of infected animals displayed lymphatic depletion, clearly visible in the lobular cortex. Similarly, in the bursa Fabricii follicles, a marked reduction of the cortical layer and a decrease in folicullar centres was revealed. A decrease in the antibody response correlated with bursa Fabricii atrophy. The clear signs of anaemia were confirmed by haematological measurements, red blood cell count, haematocrit value and haemoglobin included. On the basis of these and additional observations we propose that inoculation of duck embryos provides a suitable model for analysis of the wasting disease produced by ALV-C.

The 3' untranslated region of the chicken c-src protooncogene modulates gene expression.

Tight regulation of the Src tyrosine kinase activity is essential for a variety of cellular processes, namely transitions of the cell cycle. The peaks of Src activity are dependent on its posttranslational modifications as well as on the regulation of gene expression. The 3'UTRs of mRNAs are often crucial for rapid changes of the protein level. The chicken c-src 3'UTR effects on gene expression have been explored. The c-src 3'UTR decreased the in vivo tumorigenic potential of the src-activated mutants in chickens. This corresponds with the finding that the c-src 3'UTR reduced the Src protein and src mRNA levels and luciferase activity in vitro. Our results suggest that the chicken c-src 3'UTR plays a role in the negative control of gene expression, either transcriptionally or posttranscriptionally.

Chicken cells--oncogene transformation, immortalization and more.

Domestic chicken as a laboratory animal as well as chicken cells in vitro have been highly evaluated in several fields of experimental biology. Retrovirology and experimental oncology traditionally use this model, whose comparative aspects are still inspirative. The first (retro)viral aetiology of a tumour was recognized in the chicken and the first quantitative in vitro measurement of oncogenic transformation was developed using the chicken cells. Chicken cells (like human and primate, but unlike rodent cells) have a long primary life-span, during which they remain genetically stable. While this property is advantageous for several types of experiments, it correlates with a low propensity of the chicken cells to immortalization. Recent establishment of several continuous chicken cell lines, however, has surmounted this drawback. Furthermore, the chicken B cell line DT40 was proved to be extremely useful for gene disruption studies because of a high frequency of gene targeting not found in any vertebrate cells. In the present communication, we have tried to review several traditional achievements accomplished using the chicken model and point to newly opened areas, where chicken cells appear to be an efficient tool, particularly in cell transformation and immortalization.

Similar integration but different stability of Alus and LINEs in the human genome.

Alus and LINEs (LINE1) are widespread classes of repeats that are very unevenly distributed in the human genome. The majority of GC-poor LINEs reside in the GC-poor isochores whereas GC-rich Alus are mostly present in GC-rich isochores. The discovery that LINES and Alus share similar target site duplication and a common AT-rich insertion site specificity raised the question as to why these two families of repeats show such a different distribution in the genome. This problem was investigated here by studying the isochore distributions of subfamilies of LINES and Alus characterized by different degrees of divergence from the consensus sequences, and of Alus, LINEs and pseudogenes located on chromosomes 21 and 22. Young Alus are more frequent in the GC-poor part of the genome than old Alus. This suggests that the gradual accumulation of Alus in GC-rich isochores has occurred because of their higher stability in compositionally matching chromosomal regions. Densities of Alus and LINEs increase and decrease, respectively, with increasing GC levels, except for the telomeric regions of the analyzed chromosomes. In addition to LINEs, processed pseudogenes are also more frequent in GC-poor isochores. Finally, the present results on Alu and LINE stability/exclusion predict significant losses of Alu DNA from the GC-poor isochores during evolution, a phenomenon apparently due to negative selection against sequences that differ from the isochore composition.

DNA vaccination against v-src oncogene-induced tumours in congenic chickens.

DNA vaccination is particularly efficient for induction of cytotoxic T-lymphocyte (CTL) response. In our experiments, we used MHC(B) congenic chicken lines CB and CC (regressors and progressors of v-src-induced tumours, respectively) and a mutated, non-oncogenic v-src gene construct as the DNA vaccine. A high degree of vaccine protection against oncogenic v-src challenge was achieved in the CB line chickens. CTL response was demonstrated in vitro and by adoptive transfer of immune cells to the syngeneic host and to the CC line chickens rendered tolerant to CB cells. In the CC line chickens we observed tumour growth retardation after a low-dose DNA vaccination administered to immature recipients while higher amounts of DNA vaccine in immunocompetent chickens exerted an enhancing effect.

CpG island protects Rous sarcoma virus-derived vectors integrated into nonpermissive cells from DNA methylation and transcriptional suppression.

CpG islands are important in the protection of adjacent housekeeping genes from de novo DNA methylation and for keeping them in a transcriptionally active state. However, little is known about their capacity to protect heterologous genes and assure position-independent transcription of adjacent transgenes or retroviral vectors. To tackle this question, we have used the mouse aprt CpG island to flank a Rous sarcoma virus (RSV)-derived reporter vector and followed the transcriptional activity of integrated vectors. RSV is an avian retrovirus which does not replicate in mammalian cells because of several blocks at all levels of the replication cycle. Here we show that our RSV-derived reporter proviruses linked to the mouse aprt gene CpG island remain undermethylated and keep their transcriptional activity after stable transfection into both avian and nonpermissive mammalian cells. This effect is most likely caused by the protection from de novo methylation provided by the CpG island and not by enhancement of the promoter strength. Our results are consistent with previous finding of CpG islands in proximity to active but not inactive proviruses and support further investigation of the protection of the gene transfer vectors from DNA methylation.

Amyloid A amyloidosis in non-infected and avian leukosis virus-C persistently infected inbred ducks.

The breeding history of the first inbred strain of Khaki Campbell ducks is presented. The genetic homogeneity of this strain was tested on the basis of serum amyloid A (SAA) polymorphism and it was established that it harbours only the SAA allele A, which is expressed in liver, lung and bursa of Fabricius tissues. Pathogenic changes in control and avian leukosis virus-C (ALV-C) persistently infected ducks were evaluated during the period spanning 1 to 10 months after hatching. In both groups, AA amyloidosis was revealed and characterized. In spite of the inbred nature of animals, the incidence of amyloid A deposition varied among experiments, suggesting that additional non-genetic factors are involved. Similar variation was found in ALV-C persistently infected ducks, where only in one out of three experiments was the incidence ofAA amyloidosis significantly higher than in controls.

Retroviruses in foreign species and the problem of provirus silencing.

Retroviruses are known to integrate in the host cell genome as proviruses, and therefore they are prone to cell-mediated control at the transcriptional and posttranscriptional levels. This plays an important role especially after retrovirus heterotransmission to foreign species, but also to differentiated cells. In addition to host cell-mediated blocks in provirus expression, also so far undefined host specificities, deciding upon the pathogenic manifestation of retrovirus heterotransmission, are in play. In this respect, we discuss especially the occurrence of wasting disease and immunodeficiency syndrome, which we established also in avian species using avian leukosis virus subgroup C (ALV-C) inoculated in mid-embryogenesis in duck or chicken embryos. The problem of provirus downregulation in foreign species or in differentiated cells has been in the recent years approached experimentally. From a series of observations it became apparent that provirus downregulation is mediated by its methylation, especially in the region of proviral enhancer-promoter located in long terminal repeats (LTR). Several strategies have been devised in order to protect the provirus from methylation using LTR modification and/or introducing in the LTR sequence motifs acting as antimethylation tags. In such a way the expression of retroviruses and vectors in foreign species, as well as in differentiated cells, has been significantly improved. The complexity of the mechanisms involved in provirus downregulation and further possibilities to modulate it are discussed.

Proviral load and expression of avian leukosis viruses of subgroup C in long-term persistently infected heterologous hosts (ducks).

Proviral DNA load and expression of avian leukosis viruses of subgroup C (ALV-C) in ducks infected in mid embryogenesis were studied using quantitative PCR, RT-PCR, in situ hybridization employing ALV-specific riboprobe, and immunohistochemistry. A group of long-term surviving, non-reviremic ducks was selected for the study and compared to control reviremic animals in order to obtain information about persisting retroviruses in different duck tissues. A widespread distribution of proviruses in the tested tissues was found, but the proviral load was significantly lower in non-reviremic in comparison to reviremic animals. The only exception were brain and blood cells, in which no significant difference in the quantity of integrated proviruses was found between both categories of ducks, thus indicating an exceptional position of the brain and blood cells among all tested tissues. Contrary to reviremic, the proviruses were not transcribed in non-reviremic ducks, with the exception of brain and thymus. In the majority of non-reviremic ducks viral RNA was revealed in the brain, but no infectious virus could be recovered from this tissue. The opposite situation was observed in the thymus, where infectious virus was recovered but viral RNA remained below the detection limit of the assay. As revealed by in situ analysis, infected cells were either disseminated or focally distributed in tissues. From the long-term follow up of ALV-C in intraembryonally infected ducks we conclude that this model is suitable for the study of retrovirus persistence accompained both by the presence and absence of reviremias. The possible consequences of transmission and long-term persistence of retroviruses in the heterologous host for retroviral evolution are discussed.

Inhibition of the rous sarcoma virus long terminal repeat-driven transcription by in vitro methylation: different sensitivity in permissive chicken cells versus mammalian cells.

Rous sarcoma virus (RSV) enhancer sequences in the long terminal repeat (LTR) have previously been shown to be sensitive to CpG methylation. We report further that the high density methylation of the RSV LTR-driven chloramphenicol acetyltransferase reporter is needed for full transcriptional inhibition in chicken embryo fibroblasts and for suppression of tumorigenicity of the RSV proviral DNA in chickens. In nonpermissive mammalian cells, however, the low density methylation is sufficient for full inhibition. The time course of inhibition differs strikingly in avian and mammalian cells: although immediately inhibited in mammalian cells, the methylated RSV LTR-driven reporter is fully inhibited with a significant delay after transfection in avian cells. Moreover, transcriptional inhibition can be overridden by transfection with a high dose of the methylated reporter plasmid in chicken cells but not in hamster cells. The LTR, v-src, LTR proviral DNA is easily capable of inducing sarcomas in chickens but not in hamsters. In contrast, Moloney murine leukemia virus LTR-driven v-src induces sarcomas in hamsters with high incidence. Therefore, the repression of integrated RSV proviruses in rodent cells is directed against the LTR.

Sp1 binding sites inserted into the rous sarcoma virus long terminal repeat enhance LTR-driven gene expression.

Although the Rous sarcoma virus (RSV) long terminal repeat (LTR) is an efficient promoter of transcription, most RSV proviruses are down-regulated upon retroviral integration in non-permissive mammalian cells. Among other mechanisms, DNA methylation has been shown to be involved in proviral silencing. The presence of Sp1 binding sites has been demonstrated to be essential for protection of a CpG island and also non-island DNA regions from de novo methylation. Also, the presence of these sites in the LTRs correlates with the transcriptional activity of certain proviral structures. Using transient and stable transfection assays, we demonstrate that insertion of Sp1 binding sites into the RSV LTR remarkably increases expression of the LTR-driven genes in permissive and non-permissive cells, despite the reported negative effect of insertion of the non-specific DNA into the LTR promoter/enhancer sequences. Particular arrangement of inserted Sp1 sites was effective even in stably transfected reporter gene constructs into non-permissive mammalian cells, where additional factors exert negative effects on expression.

The LTR, v-src, LTR provirus in H-19 hamster tumor cell line is integrated adjacent to the negative regulatory region.

The tumor hamster cell line H-19 harbors a single copy LTR, v-src, LTR provirus that becomes permanently transcriptionally suppressed in morphological revertants segregating at high rate from this cell line. Our previous data document that the provirus suppression is mediated by epigenetic cell-regulatory mechanisms. In this report, we concentrate on cellular sequences neighboring the integration site. The locus is unique for Syrian hamster and is not detectable in DNA of several animal species. No restriction sites that usually hint at the presence of CpG islands were found in the significantly close vicinity of the provirus. Nevertheless, the chromosomal DNA flanking the provirus is rich in GC content (57.8%). We localized a 0.5-kb region downstream from the provirus that remarkably inhibits transcription in the transient expression assay and is effective both on the homologous RSV LTR promoter/enhancer and heterologous SV40 promoter. We propose that a cellular trans-acting factor is involved in the silencing of the reporter gene. Since this activity is comparable both in transformed and revertant cells, we speculate that this down-regulatory region makes the permissive integration locus prone to provirus silencing initiated by other fluctuating stimuli.

Frequent detection of reviraemia in ducks persistently infected with avian leukosis retroviruses.

Ducks intraembryonally infected with avian leukosis viruses of subgroup C (ALV-C) were followed for a long period (up to 6.8 years), and the viraemia and production of virus-neutralizing antibodies were measured. In three independent experiments comprising ducks inoculated with uncloned and/or molecularly cloned ALV-C, we found that after the elimination of primary post-hatching viraemia, reviraemia could be detected in 60-70% of infected animals. Based on the course of viraemia, the individual ducks were assigned to four different groups: Group I (no reviraemia), Group II (one transient reviraemic period), Group III (one persistent reviraemic period), Group IV (fluctuating reviraemia). In comparison to sera from ducks included in Group I and/or II, a significant decrease in neutralizing activity of sera from animals comprised in Group III and/or IV was observed. Two out of four reviraemic viruses were not neutralized by antiserum against ALV-C, instead their infectivity was enhanced. Long-term follow-up of the cell-associated virus revealed that its rescuability by cocultivation with chicken embryo fibroblasts fluctuated in about 50% of animals. In the reviraemic phase of infection, integrated proviruses could be detected by Southern blotting in a majority of tissues examined. Our data document that many features recognized in lentiviruses are valid also for oncoviruses transmitted to heterologous hosts and substantiate further the suitability of ALV-C-infected ducks as a model for studying persistent retroviral infection.

High rate of morphological reversion in tumor cell line H-19 associated with permanent transcriptional suppression of the LTR, v-src, LTR provirus.

The highly malignant line of morphologically transformed H-19 hamster tumor cells that harbor a single LTR, v-src, LTR provirus segregates morphologically flat revertants at the rate of 1.4 to 2.4 x 10(-3)/cell/cycle. Revertants behave like almost nonmalignant cells; they keep the provirus within an unaltered junction DNA fragment. However, the provirus is methylated, permanently transcriptionally silent, and not rescuable. Using the polymerase chain reaction, we have synthesized the whole proviral structure from two revertants and established that the left-hand long terminal repeats assuring transcription remained structurally intact. Moreover, the cloned proviral DNAs from three revertants were shown to produce tumors in chickens. The unusually high reversion rate together with the finding of structural integrity of proviral transcriptional signals in revertants indicate strongly that the reversion has been mediated by cell-regulatory mechanisms.

src-specific immunity in inbred chickens bearing v-src DNA- and RSV-induced tumors.

The growth pattern (progression/regression) of v-src DNA- and Rous sarcoma virus (RSV)-induced tumors was analogous on a panel of inbred chicken lines. The decisive role of the major histocompatibility complex [Mhc(B)] alleles in resistance to the progression of these tumors was formally proved in segregating backcross populations. The immune mechanism of tumor regression was demonstrated by both in vivo and in vitro assays. A protective effect of v-src-specific immunity against RSV challenge was shown in Rous sarcoma regressor, line CB (B12/B12). Immune cells from regressors of v-src DNA-induced tumors can protect syngeneic hosts from the development of tumor after challenge with both v-src DNA and RSV. Suppression of RSV-induced tumor cell growth in vitro was also achieved by the use of cocultivation with spleen cells from chickens in which v-src DNA-induced tumors had regressed. This in vitro sarcoma-specific response was Mhc(B)-restricted. Chickens of the congenic Rous sarcoma progressor line CC (B4/B4) are sometimes able to regress v-src DNA-induced tumors, but immune cells can only slow the growth of v-src DNA-induced tumors in syngeneic hosts. This suggests that the primary reason for the susceptibility of CC chickens is a weak v-src-specific immune response. Furthermore, some of the v-src DNA-induced tumors were transplantable across the Mhc(B) barrier. The growth of tumor allografts was able to be facilitated when immunological tolerance to the B-F/L region antigens (class I and class II) had been established. This demonstrated that a high tumorigenicity of the transplantable tumor was not due to the lack of Mhc(B) antigens on tumor cells.